Bottom Line:
After extraction of the kettin-associated actin, the A-band edges were also stained.Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin.We conclude that kettin is attached not only to actin but also to the end of the thick filament.

Affiliation: Institute of Physiology and Pathophysiology, University of Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACTKettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and alpha-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM-I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with mu-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin- mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms.

fig5: Effect of treatment with μ-calpain (A–E) and Igase (F–H) on Drosophila IFM myofibrils. (A) IF of single myofibril stained with α-kettin Ig16 after calpain treatment and stretch; the A-band edge is labeled (arrowheads), and the Z-disc is labeled also in some sarcomeres (arrow). (B) α-Projectin staining of stretched sarcomeres showed a fuzzy epitope at the A-band edge (arrowheads) and also Z-disc labeling (arrow). (C) PEVK sequence was not stained by 9D10 antibody in calpain-treated, stretched, myofibrils. (D) Low percentage SDS-gel to show the effect of μ-calpain (3 μg/ml, 25°C, 45 min) on high molecular weight proteins. Only kettin (K) isoforms are substantially digested, whereas projectin (P) and M-line protein (M) remain largely intact. Nebulin (N) and titin (T) from rabbit soleus muscle are used as standards. (E) Lower molecular weight proteins are not affected by μ-calpain treatment as detectable on 10–18% SDS-gradient gels. (F) Igase-mediated digestion of troponin H effectively decreased the staining intensity of α-TnH34 antibodies (MAC 143) on myofibrils. (G) 12% SDS-gel electrophoresis of washed IFM myofibrils to separate TnH33 and TnH34 isoforms. Igase treatment eliminated the TnH34 isoform. (H) Western blot with antibodies to either TnH33 or TnH34 confirms that Igase treatment removed TnH34 but left TnH33 intact.

Mentions:
A Ca2+-dependent protease, μ-calpain (3 μg/ml), selectively digests kettin in Drosophila IFM sarcomeres (Fig. 5, D and E) . After treatment of IFM myofibrils with μ-calpain, SL homogeneity upon stretch in relaxing buffer was much improved compared with control myofibrils (Fig. 3 C). More importantly, rhodamine-phalloidin staining revealed that the actin filaments could now be pulled out of the A-bands. Only rarely did actin still break at the Z-disc (Fig. 3 C, arrow); usually, the actin filaments of two adjacent half sarcomeres remained connected at the Z-band (Fig. 3 C, arrowhead at bottom). Thus, cleavage of kettin removed the elements that normally prevent Drosophila IFM sarcomeres from tolerating larger stretches. The leftover sarcomere structure might be held together by the remaining projectin molecules. (Projectin is cleaved only slightly at the μ-calpain concentration of 3 μg/ml chosen [Fig. 5 D].)

fig5: Effect of treatment with μ-calpain (A–E) and Igase (F–H) on Drosophila IFM myofibrils. (A) IF of single myofibril stained with α-kettin Ig16 after calpain treatment and stretch; the A-band edge is labeled (arrowheads), and the Z-disc is labeled also in some sarcomeres (arrow). (B) α-Projectin staining of stretched sarcomeres showed a fuzzy epitope at the A-band edge (arrowheads) and also Z-disc labeling (arrow). (C) PEVK sequence was not stained by 9D10 antibody in calpain-treated, stretched, myofibrils. (D) Low percentage SDS-gel to show the effect of μ-calpain (3 μg/ml, 25°C, 45 min) on high molecular weight proteins. Only kettin (K) isoforms are substantially digested, whereas projectin (P) and M-line protein (M) remain largely intact. Nebulin (N) and titin (T) from rabbit soleus muscle are used as standards. (E) Lower molecular weight proteins are not affected by μ-calpain treatment as detectable on 10–18% SDS-gradient gels. (F) Igase-mediated digestion of troponin H effectively decreased the staining intensity of α-TnH34 antibodies (MAC 143) on myofibrils. (G) 12% SDS-gel electrophoresis of washed IFM myofibrils to separate TnH33 and TnH34 isoforms. Igase treatment eliminated the TnH34 isoform. (H) Western blot with antibodies to either TnH33 or TnH34 confirms that Igase treatment removed TnH34 but left TnH33 intact.

Mentions:
A Ca2+-dependent protease, μ-calpain (3 μg/ml), selectively digests kettin in Drosophila IFM sarcomeres (Fig. 5, D and E) . After treatment of IFM myofibrils with μ-calpain, SL homogeneity upon stretch in relaxing buffer was much improved compared with control myofibrils (Fig. 3 C). More importantly, rhodamine-phalloidin staining revealed that the actin filaments could now be pulled out of the A-bands. Only rarely did actin still break at the Z-disc (Fig. 3 C, arrow); usually, the actin filaments of two adjacent half sarcomeres remained connected at the Z-band (Fig. 3 C, arrowhead at bottom). Thus, cleavage of kettin removed the elements that normally prevent Drosophila IFM sarcomeres from tolerating larger stretches. The leftover sarcomere structure might be held together by the remaining projectin molecules. (Projectin is cleaved only slightly at the μ-calpain concentration of 3 μg/ml chosen [Fig. 5 D].)

Bottom Line:
After extraction of the kettin-associated actin, the A-band edges were also stained.Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin.We conclude that kettin is attached not only to actin but also to the end of the thick filament.

Affiliation:
Institute of Physiology and Pathophysiology, University of Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACTKettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and alpha-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM-I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with mu-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin- mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms.